Isolation and chemical composition of the leucocytes from donkey, horse, mule and pig.

1. Leucocytes from the blood of adult and young donkeys (Equus asinus L.), adult horses (Equus caballus L.), adult mules (Equus asinus x Equus caballus) and adult pigs (Sus scrofa L.) were obtained in a high degree of purity (99.9%) using Na2-EDTA-dextrans mixtures. 2. Sialic acids were released, purified, identified and determined from both non-delipided and delipided leucocytes. 3. N-glycolylneuraminic was the predominant sialic acid. N-acetylneuraminic acid and N,O-diacetyl-neuraminic acid were also found in all materials. Except in pig, other unidentified sialic acid(s) were also detected. 4. The concentration of total sialic acids (microgram/mg protein) is different according to the species, and in donkey species according to the age. 5. Galactose, glucose, mannose, fucose and (in a less amount) ribose were determined. Their total content is about 2-3-fold that of hexosamines. 6. There is a higher cholesterol content in adult donkey leucocytes than in those of young ones. 7. Total lipids, cholesterol or phospholipid contents are similar among the leucocytes of the above-mentioned species. 8. The similarities are marked in the electrophoresis patterns of proteins and glycoproteins for the donkey, mule and horse samples. 9. The molecular weights for leucocytes proteins were estimated as ranging between 230,000 and 20,000; and for the main protein bands, between 120,000 and 22,000.     

Evolutionary movement of centromeres in horse, donkey, and zebra

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years).These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.     

Glycosylation at the fetomaternal interface: does the glycocode play a critical role in implantation?

During pregnancy, the heavily glycosylated surfaces of the implanting blastocyst and maternal uterine epithelium interact in a highly controlled and specific manner. Examination of this interface in species that show interdigitation of embryonic and maternal surfaces (epitheliochorial placentation) shows that each has its own particular pattern of glycosylation or glycotype, and that closely related and/or interbreeding species e.g. horse and donkey or llama and guanaco, have very similar glycotypes. Implantation of interspecies hybrids is facilitated, when the blastocyst has an outer cell layer bearing glycans that are compatible with the maternal host. We refer to this mutual compatibility as a glycocode. The probability that hybrid embryo glycotypes differ from those normally associated with the host species may account for the high pregnancy failure rates seen in interspecies breeding. We suggest the maternal host selects between genotypically distinct embryos, and this selection depends partly on cell surface glycosylation. We infer that the glycocode plays a critical role in implantation, for if the survival of modified genotypes results in fitter offspring with altered placental glycosylation, selection pressure downstream may in turn act to drive adaptations in the maternal surface glycotype to produce a complementary glycocode, thus leading eventually to the creation of new species. We speculate that glycan microheterogeneity plays a specific role in this process.     

Comparative studies on blood serum alpha-L-fucosidases from several mammalian species.

1. Peripheral blood serum alpha-L-fucosidases have been studied from various mammalian species: Sus scropha var domestica L. (pig), Capra hircus L. (goat), Bos taurus L. (bull, races Morucha and Charolais), Equus caballus L. (horse) and Equus asinus L. (donkey). 2. Fluorimetric and spectrophotometric procedures were used for determination of alpha-L-fucosidases. 3. alpha-L-Fucosidases were more active towards fluorescent substrates than towards chromogenic substrates. 4. pH optima values of the enzymes are: (A) 5.5 for sera from all above-mentioned species when fluorescent substrates were employed; (B) 4.0 for goat, 4.5 for bull, 5.0 for pig and 4.5-5.0 for horse and donkey sera when chromogenic substrates were used. 5. pH activity profiles are very similar for two races (Morucha and Charolais) of the same species (Bos taurus L.) and also for two species of the same genus (Equus caballus and Equus asinus L.). 6. These serum alpha-L-fucosidases are very labile under heat treatment, even at 37 degrees C.     

Horse domestication and conservation genetics of Przewalski's horse inferred from sex chromosomal and autosomal sequences

Despite their ability to interbreed and produce fertile offspring,there is continued disagreement about the genetic relationshipof the domestic horse (Equus caballus) to its endangered wildrelative, Przewalski's horse (Equus przewalskii). Analyses havediffered as to whether or not Przewalski's horse is placed phylogeneticallyas a separate sister group to domestic horses. Because Przewalski'shorse and domestic horse are so closely related, genetic datacan also be used to infer domestication-specific differencesbetween the two. To investigate the genetic relationship ofPrzewalski's horse to the domestic horse and to address whetherevolution of the domestic horse is driven by males or females,five homologous introns (a total of 3 kb) were sequenced onthe X and Y chromosomes in two Przewalski's horses and threebreeds of domestic horses: Arabian horse, Mongolian domestichorse, and Dartmoor pony. Five autosomal introns (a total of6 kb) were sequenced for these horses as well. The sequencesof sex chromosomal and autosomal introns were used to determinenucleotide diversity and the forces driving evolution in thesespecies. As a result, X chromosomal and autosomal data do notplace Przewalski's horses in a separate clade within phylogenetictrees for horses, suggesting a close relationship between domesticand Przewalski's horses. It was also found that there was alack of nucleotide diversity on the Y chromosome and highernucleotide diversity than expected on the X chromosome in domestichorses as compared with the Y chromosome and autosomes. Thissupports the hypothesis that very few male horses along withnumerous female horses founded the various domestic horse breeds.Patterns of nucleotide diversity among different types of chromosomeswere distinct for Przewalski's in contrast to domestic horses,supporting unique evolutionary histories of the two species.     

Mass spectral analysis of domestic and wild equine apoA-I and A-II: detection of unique dimeric forms of apoA-II

In pigs, humans, chimpanzees and probably other great apes, a cysteine at residue 6 enables apolipoprotein A-II to form a homodimer. However, the apoA-IIs of other primates, lacking a cysteine residue, are monomeric. We have already reported that horse apoA-IIs form homodimers due also to a cysteine at residue 6. In this study, we wanted to determine whether other equine apoA-IIs might be monomeric. The high density lipoproteins were ultracentrifugally isolated from the plasmas of a horse (Equus caballus), a donkey (Equus asinus) and five wild equines: two types of zebras (Equus zebra hartmannae and Equus zebra quagga boehmi), a Przewalski's horse (Equus przewalskii), a Somali ass (Equus africanus somalicus) and a kiang (Equus kiang holdereri). Using liquid chromatography with electrospray-ionization mass spectrometry, we were able to obtain accurate values for the molecular masses of apoA-I and apoA-II. Homodimeric apoA-IIs were observed in each of the animals studied. The donkey had unique dimers, consisting of the proapolipoprotein A-II linked by a disulfide bond either to a mature apoA-II monomer or another proapoA-II. In addition, our data indicate that small amounts of apoA-I and apoA-II apparently are acylated.     

Donkey and horse alpha 1 B-glycoprotein: partial characterization and new alleles.

1. The donkey postalbumin protein has been shown to be the equivalent of human alpha 1 B-glycoprotein by protein immunoblotting and N-terminal amino acid sequence. 2. The horse A1B system (already identified as the homologue of human alpha 1 B-glycoprotein) and the donkey alpha 1 B-glycoprotein were characterized further for terminal sialic acid content, isoelectric point, amino acid composition and affinity for the dye-ligand, Cibacron Blue F3GA (known to bind human alpha 1 B-glycoprotein). 3. Two new alleles in the horse A1B system were found, bringing the total number of alleles to five. No polymorphism was found in the donkey alpha 1 B system. 4. As expected the first 20 N-terminal residues of the donkey and horse proteins are highly conserved with only two differences being found. 5. The polymorphism of the horse alpha 1 B-glycoproteins may be due in part to differing numbers of terminal sialic acid residues and the higher electrophoretic mobility of the donkey alpha 1 B-glycoprotein may be due in part to increased sialylation. 6. The horse and donkey alpha 1 B-glycoproteins exhibited differences in affinity for the dye-ligand, Cibacron Blue F3GA, with the donkey alpha 1 B-glycoprotein not being bound.     

Oestrogen production by the preimplantation donkey conceptus compared with that of the horse and the effect of between-species embryo transfer

Aromatase distribution in membranes of preimplantation horse and donkey conceptuses was compared by measuring the incorporation of [3H]androstenedione into oestrone and oestradiol-17 beta. In the donkey conceptus, aromatase activity was similar in all the tissues examined (yolk sac, chorionic girdle and allantochorion), whereas in the horse it was generally lower and showed the relationship chorionic girdle greater than yolk sac greater than allantochorion. A higher proportion of labelled precursor was incorporated into oestradiol-17 beta by extra-embryonic tissues of the donkey compared with those of the horse. In contrast to previous results, aromatase in the chorionic girdle did not decline progressively before its migration into the endometrium on Day 36 to form the endometrial cups. The chorionic girdle of a donkey conceptus carried in the uterus of a mare failed to invade the surrogate horse endometrium and aromatase activity was still high in this tissue at Day 42. Aromatase distribution in 2 transferred donkey-in-horse conceptuses resembled that of the fetal, rather than the maternal, genotype indicating a lack of effect of the maternal environment.     

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis

Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis.     

Nutrition of bison (<Emphasis Type="Italic">Bison bison</Emphasis>), camels (<Emphasis Type="Italic">Сamelus bactrianus</Emphasis>), and horses (<Emphasis Type="Italic">Equus caballus</Emphasis>) from their joint grazing on an isolated steppe pasture

The nutrition of free-ranging bison (Bison bison), domestic horses (Equus caballus), and camels (Camelus bactrianus) were investigated from their joint grazing on a forb-grass steppe pasture. The species composition of consumed plants, selectivity of food plants, and digestibility of plant forage were assessed. Species of the plants consumed and their share in the diet have been determined using microhistological analysis of feces. The plant food selectivity (FS) was estimated by the ratio of the share of plant species in the diet of animals to their portion in the plant community of the pasture. The digestibility is calculated by the ratio of inert (undigested) components (silicon, lignin) in the diet and feces. In summer (June), these characteristics were similar for horses and bison. Both species are typical animals consuming gramineous plants: the share of these plants in their diet is 81–83%, their selectivity of graminoids is equal (1.4), and the digestibility of food is similar (49–51%). Camels differed from bison and horses by all food characteristics: forbs (mainly ruderal annual Bassia sedoides (43%) and Atriplex tatarica (20%)) predominated at 86%; graminoids amounted to 14%. The food selectivity coefficient (FS) for forbs was 2.1, including 7.7 and 2.9 for Bassia and Atriplex, respectively. The digestibility coefficient (60%) was much higher for camels in comparison with bison and horses. Under joint grazing, species with different forage selectivity (horse–camel or bison–camel) affect the plant community of a pasture evenly and preserve its species diversity.     

Sialic acid in hemolymph and affinity purified lectins from two marine bivalves

Sialic acids have been implicated in a variety of complex biological regulatory and signalling events and their functional importance is reflected by their presence in a wide variety of phyla. Potentially they may inhibit intermolecular and intercellular interactions. Lectins that exhibit specificity for sialic acid or sialoglycoconjugates are ubiquitous in the body fluids of invertebrates and this has supported the assumption that these lectins are involved in defense against microbes that express sialic acids on their surfaces. This biological function has also been inferred from the absence of sialic acids in lower invertebrates. However, most invertebrate lectins are heterogeneous and may also bind other ligands. The biological significance of the different carbohydrate specificities are not yet known. We have demonstrated the presence of sialic acids in hemolymph from two marine bivalves, the Pacific oyster Crassostrea gigas (≈15 μg ml⁻¹) and the horse mussel Modiolus modiolus (48–100 μg ml⁻¹) by several different assays. The sialic acid was mostly in free form. Affinity purified lectins from the horse mussel also contained bound sialic acids (2–5 μmol g⁻¹). Oyster hemolymph stimulated the in vitro phagocytosis of bacteria by oyster hemocytes. The stimulation by hemolymph is facilitated by a dialyzable component, that apparently is active irrespective of the binding to sialic acid (BSM). Addition of sialic acid had no significant effect on the in vitro phagocytosis of bacteria by oyster hemocytes.     

Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa

The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances.     

Evolution of the placenta during the early radiation of placental mammals

The chorioallantoic placenta is an organ of gaseous exchange that exhibits a high degree of structural diversity. One factor determining oxygen transfer across the placenta, the diffusion distance, is in part dependent on the number of cell layers separating maternal from fetal blood. This interhaemal barrier occurs in three principal variants. The focus of this review is on determining how the barrier evolved in placental mammals. The analysis was based on current knowledge of placental structure, as far as possible using ultrastructural data, and on current views about the evolution of placental mammals, derived from molecular phylogenetics. We show that epitheliochorial placentation, the least invasive type, is a derived state and discuss factors that may have determined its evolution with reference to conflict theory, as applied to the allocation of resources between mother and fetus. It is not yet possible to determine which of the two more invasive types of placentation occurred in the last common ancestor of crown placentals. Depending on tree topology and taxon sampling, the result achieved is either endotheliochorial, haemochorial or unresolved. Finally we discuss other factors important to placental gas exchange and point to physiological variables that might become amenable to phylogenetic analysis.     

Interspecific variation of zona pellucida glycoconjugates in several species of marsupial

The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O-acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide beta-galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O-acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of beta-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O-acetylation of sialic acids on the internal compartment of the zonae pellucidae.     

Chromosomal distribution of the telomere sequence (TTAGGG)(n) in the Equidae

Telomeres are a class of repetitive DNA sequences that are located at chromosome termini and that act to stabilize the chromosome ends. The rapid karyotypic evolution of the genus Equus has given rise to ten taxa, all with different diploid chromosome numbers. Using fluorescence in situ hybridization (FISH) we localized the mammalian telomere sequence, (TTAGGG)(n), to the chromosomes of nine equid taxa. TTAGGG signal was located at chromosome termini in all species, however additional signal was seen at interstitial sites on some chromosomes in the Burchell's zebra, Equus quagga burchelli, the Hartmann's zebra, Equus zebra hartmannae, and at large heterochromatin-associated regions on the chromosomes of the donkey, Equus asinus. The interstitial signal in the zebras may be a relic of an ancient telomere-telomere fusion and mark the point at which two ancestral chromosomes may have fused. For the donkey, the heterochromatin-associated signal may represent degenerate telomere-like satellite sequences and identify a second type of satellite DNA for this taxon.     

Molecular cloning and sequence analysis of prion protein gene in Xiji donkey in China

Prion diseases are a group of human and animal neurodegenerative disorders caused by the deposition of an abnormal isoform prion protein (PrP^Sc) encoded by a single copy prion protein gene (PRNP). Prion disease has been reported in many herbivores but not in Equus and the species barrier might be playing a role in resistance of these species to the disease. Therefore, analysis of genotype of prion protein (PrP) in these species may help understand the transmission of the disease. Xiji donkey is a rare species of Equus not widely reared in Ningxia, China, for service, food and medicine, but its PRNP has not been studied. Based on the reported PrP sequence in GenBank we designed primers and amplified, cloned and sequenced the PRNP of Xiji donkey. The sequence analysis showed that the Xiji donkey PRNP was consisted of an open reading frame of 768 nucleotides encoding 256 amino acids. Amino acid residues unique to donkey as compared with some Equus animals, mink, cow, sheep, human, dog, sika deer, rabbit and hamster were identified. The results showed that the amino acid sequence of Xiji donkey PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of other Equus species in this study. Amino acid sequence analysis showed high identity within species and close relation to the PRNP of sika deer, sheep, dog, camel, cow, mink, rabbit and hamster with 83.1–99.7% identity. The results provided the PRNP data for an additional Equus species, which should be useful to the study of the prion disease pathogenesis, resistance and cross species transmission.     

Comparative mapping in equids: the asine X chromosome is rearranged compared to horse and Hartmann's mountain zebra

The X chromosomes of the extant equids, in general, share morphology and banding pattern similarities. However, the donkey X is, in part, an exception because of significantly different centromeric index and variant banding patterns in the pericentromeric region. To verify the underlying molecular basis of this difference, twelve equine BAC clones were FISH mapped to donkey (EAS) and Hartmann's mountain zebra (EZH) metaphase spreads. Loci from the terminal region of Xp and distal to terminal regions of the Xq showed the same order and relative position in all three species, implying cross-species conservation of these chromosomal segments. However, loci from the proximal/pericentromeric regions of either arms showed similar FISH locations in horse and zebra but a slightly deviant location and relative position in the donkey. Three of the markers (tel-OTC, TRAP170 and (ps)ALDH2- cen) located on the short arm of ECAX and EZHX were found inverted on the long arm of EASX, along with the transposition of the centromere. This molecular evidence of a pericentromeric inversion helps define the likely evolutionary breakpoints causing the rearrangement. The breakpoints most likely correspond to the region between Xp16-->q12 in the horse and Xp12-->q13 in the donkey. The findings coupled with the highly conserved X-chromosome gene order between horse and outgroup species, human and cat, suggest that the equine type X is ancestral while the asine type X arose as a result of an independent inversion event. The study adds two new markers to horse, 11 to donkey and 12 to Hartmann's zebra gene maps, thus contributing to the expansion of comparative maps in the equids.     

Comparative electron microscopy of chorio-allantoic placental barrier in some Indian Chiroptera

In the present study the comparative ultrastructure of the definitive chorio-allantoic placental barrier has been studied in considerable detail in six species of bats, representing six different families and both suborders of Chiroptera, by electron microscopy, and these species illustrate different kinds of interhaemal membranes met with among bats. The definitive chorio-allantoic placenta of Rousettus leschenaulti is haemodichorial, since the syncytiotrophoblast and cytotrophoblast layers are present to term. The fine structure of the placental barrier in the labyrinth of the definitive placenta of Rhinopoma hardwickei hardwickei is essentially endotheliomonochorial due to the presence of a single layer of cytotrophoblast and maternal endothelial cells. The placenta of Taphozous melanopogon, examined electron-microscopically in the present study, shows a thick maternal endothelium, a continuous interstitial membrane and the presence of a single layer of syncytiotrophoblast. The placenta of Megaderma comprises a typical endotheliochorial labyrinth and the presence of two layers of trophoblast. In Rhinolophus rouxi, the mature placenta during advanced pregnancy resembles that of Megaderma, its labyrinth containing large maternal capillaries with maternal endothelial cells and the two layers of trophoblast. Finally, the placental barrier of Hipposideros fulvus fulvus is haemodichorial due to the presence of two layers of trophoblast and the absence of maternal endothelial cells.